UvrC Coordinates an O₂-Sensitive [4Fe4S] Cofactor

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinorganic Chemistry

Background:

• [4Fe4S] clusters are vital cofactors in enzymes across all life domains.
• Observing these clusters in nucleic acid processing enzymes is difficult due to weak signatures and cofactor lability.
• UvrC, a bacterial nucleotide excision repair (NER) endonuclease, was investigated for [4Fe4S] cluster involvement.

Purpose of the Study:

• To investigate the presence and role of a [4Fe4S] cluster in the UvrC enzyme.
• To characterize the cofactor's stability and its impact on UvrC's DNA binding affinity and function.
• To determine the redox activity of the [4Fe4S] cluster within UvrC.

Main Methods:

• Sequence alignments and anaerobic purification techniques.
• Iron quantification, UV-visible, and electron paramagnetic resonance (EPR) spectroscopies.
• DNA binding assays and DNA electrochemistry.

Main Results:

• UvrC exhibits characteristics consistent with [4Fe4S] cluster coordination (60-70% incorporation).
• The [4Fe4S] cluster in UvrC is susceptible to oxidative degradation.
• Holo-UvrC displays high-affinity DNA binding (80-100 nM) compared to apo-UvrC.
• The [4Fe4S] cluster is redox-active and participates in DNA-mediated charge transport (midpoint potential of 90 mV vs NHE).

Conclusions:

• The [4Fe4S] cluster is critical for UvrC function, enhancing its DNA binding affinity.
• The cofactor's redox activity suggests a role in DNA repair mechanisms beyond simple binding.
• Understanding the [4Fe4S] cluster in UvrC provides insights into DNA repair pathways and metalloenzyme function.